Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration.

Nitric oxide (NO), produced by distinct nitric oxide synthase (NOS) isoforms, and prostaglandins generated by expression of cyclooxygenases are important mediators in tumor progression. Previous studies have shown that NO can influence the formation of prostaglandin E2 (PGE2). We provide evidence that NO, derived from iNOS and eNOS activity in LMM3 murine mammary adenocarcinoma cell line, is involved in tumor angiogenesis and in tumor cell migration. LMM3 cells that also stimulate their neovascularization activity and migration liberate high basal amounts of PGE2. There is large amount of evidence that postulates positive regulatory interactions between NOS and cyclooxygenase (COX) isoforms. We here show that, in the LMM3 cell line, while PGE2 exerts a positive modulation on NOS activity, NO closes the loop with a negative feed back on COX activity. We also provide evidence of a positive regulatory effect of protein tyrosine kinases on NOS as well as on COX enzymatic functions affecting tumor induced angiogenesis and cell migration.

Characterization of a fibroblastoid mammary carcinoma cell line (LM2) originated from a mouse adenocarcinoma.

We established and characterized a new mammary tumor cell line, LM2, derived from M2 mammary adenocarcinoma which spontaneously appeared in a BALB/c female mouse. The LM2 cell line has been maintained in culture for more than 40 passages and grows as poorly differentiated elongated cells. Ultrastructural and immunocytochemistry analysis revealed characteristic features of adenocarcinoma. Cytogenetic studies showed that LM2 cells are fundamentally hypotetraploid. They express metalloproteinases (MMP) and show high levels of plasminogen activator type urokinase (uPA). They were sensitive to nitric oxide (NO)-mediated cytotoxicity when NO derived from an exogenous donor. In vivo, although LM2 cells were able to grow in the lungs, they could not metastasize to the same target organ from s.c. primary tumors. The LM2 mouse mammary adenocarcinoma cell line is a suitable model to examine different aspects of tumor biology, in particular those related to the different pathways involved in the metastatic cascade and in the cytotoxicity mediated by NO.

Differential nitric oxide release and sensitivity to injury in different murine mammary tumor cell lines.

The purpose of this study was to determine whether nitric oxide (NO) production by different mammary tumor cell lines correlated with their sensitivity to NO mediated injury. Three mammary tumor cell lines LM2, LM3 and LMM3 syngeneic to BALB/c mice were cultured in vitro with IFNgamma + LPS. Different levels of NO production among the three lines were detected in culture supernatants. The only tumor cell line which did not produce NO (LM2) showed the highest sensitivity to SNP-derived NO cytotoxicity (87%), while LM3 and LMM3 which both produced higher levels of NO than LM2, showed lower cytotoxicity by SNP (39% and 22% respectively). Spleen cells (SC) from M2 tumor bearing mice (TBM) were able to lyse LM2 cells by NO-dependent mechanisms. SC from M3-TBM exerted cytotoxicity against LM3 cells mainly by NO-independent mechanisms. Thus, we postulate an inverse correlation between NO production and NO mediated cytotoxicity in the three mammary tumor cell lines. It is possible that tumor cells producing NO develop mechanisms to resist NO injury.

Lymphocyte-induced angiogenesis: effect of different antigenic stimuli.

It is known that tumor cells activate spleen cells to induce an angiogenic response. In this report we studied whether different antigenic stimuli, other than tumor cells, were able to activate spleen lymphocytes to induce angiogenesis in syngeneic combination (SLIA). For this purpose, mice were inoculated with sheep red blood cells (SRBC), allogeneic kidney and syngeneic fetal tissues. The effect of pregnancy (syngeneic or allogeneic) on the ability of spleen cells to induce a neovascular response was also assessed. None of the different stimuli were able to induce spleen lymphocytes to evoke angiogenesis. Although allogeneic lymphocytes from virgin females induced a strong neovascular response, the same population, but from allogeneic pregnant mice, did not evoke this response. We conclude that tumor cells seem to be the only antigenic stimuli able to activate spleen lymphocytes to induce SLIA.

Polyamines prevent DFMO-mediated inhibition of angiogenesis.

Tumor growth mainly depend on formation of new blood vessels. DFMO (alpha-difluoromethylornithine), an inhibitor of polyamine biosynthesis, inhibits tumor growth in many animal tumors. Our investigation was to evaluate the requirement of polyamines for induction of angiogenesis by tumor cells and spleen lymphocytes from tumor-bearing mice. In this regard, we have added DFMO to cell cultures. The neovascular response induced either by tumor cells or spleen lymphocytes was completely abrogated. This inhibition could be reversed by the addition of exogenous putrescine. These findings suggest that the effect of DFMO on angiogenesis is, in part, mediated by the inhibition of polyamine biosynthesis.

Immunomodulation by soluble factors from tumor cells cultured in vivo in diffusion chambers.

The delayed-type hypersensitivity (DTH) response and lymphocyte-mediated angiogenesis were determined in mice bearing in vivo cultures of mammary tumor cells in diffusion chambers (DCs). Soluble tumor products which diffuse from the DCs were able to stimulate the immune system for both the DTH reaction and angiogenic activity by spleen cells.

Isolated soluble fractions from spleen cell culture supernatants induce tumor enhancement.

Supernatants of spleen cell culture from small-tumor-bearing mice, large-tumor-bearing mice, and large-tumor resected mice were fractionated by Sephadex G-100. The biological activity on tumor growth of all fractions was tested in vivo. It was found that only one fraction (MW: 220-250 kD) from spleen cell supernatants of small-tumor-bearing mice or large-tumor resected mice enhanced tumor growth. In spleen cell supernatants from large-tumor-bearing mice, two fractions had enhancing activity (MW: 220-250 and 100-10 kD). Thus, the surgical resection of large tumor induced disappearance of enhancing activity from the fraction of lower molecular weight.

[Immunoregulation of tumor growth in a murine model]. / Inmunorregulación del crecimiento tumoral en un sistema murino.

Different spleen and tumor cell factors modifying tumoral and metastatic growth were studied. Spleen cell culture supernatants (SCS) from small and large tumor-bearing mice enhanced tumor growth. After tumor surgery, tumor enhancement was only mediated by supernatants from large tumor resected mice. Tumor facilitation and angiogenic response were mediated by the same supernatants; different fractions for these two activities were characterized. T and non-T cells, depending on tumor burden, were responsible for the enhancing activity; but angiogenesis depended only on T cells. While augmentation of metastatic spread was produced by tumor antigens (soluble tumor extracts, tumor-cell supernatants, formolized tumor cells), primary tumor development was not modified by tumor-cell supernatants. Increased incidence of metastases was also mediated by SCS from tumor resected mice which had previously been inoculated with tumor antigens. Immune status of tumor-resected mice was evaluated by delayed-type hypersensitivity reaction. Tumor cell membranes enriched with cholesterol-hemisuccinate were able to increase anti-tumor immune response.

Inmunorregulación del crecimiento tumoral en un sistema murino. / [Immunoregulation of tumor growth in a murine model]

Different spleen and tumor cell factors modifying tumoral and metastatic growth were studied. Spleen cell culture supernatants (SCS) from small and large tumor-bearing mice enhanced tumor growth. After tumor surgery, tumor enhancement was only mediated by supernatants from large tumor resected mice. Tumor facilitation and angiogenic response were mediated by the same supernatants; different fractions for these two activities were characterized. T and non-T cells, depending on tumor burden, were responsible for the enhancing activity; but angiogenesis depended only on T cells. While augmentation of metastatic spread was produced by tumor antigens (soluble tumor extracts, tumor-cell supernatants, formolized tumor cells), primary tumor development was not modified by tumor-cell supernatants. Increased incidence of metastases was also mediated by SCS from tumor resected mice which had previously been inoculated with tumor antigens. Immune status of tumor-resected mice was evaluated by delayed-type hypersensitivity reaction. Tumor cell membranes enriched with cholesterol-hemisuccinate were able to increase anti-tumor immune response.

Angiogenic activity by spleen cell supernatants from tumor-bearing and tumor-resected mice.

Supernatants of cultured spleen cells (SCS) from small tumor-bearing mice (STBM) and large tumor-bearing mice (LTBM) are able to enhance tumor growth as well as accelerate tumor takes in vivo when inoculated 24 h before tumor implant. After tumor resection enhancing activity disappears at a rate depending on the size of the resected tumor. Spleen cells from tumor-bearing mice also evoke a complex vascular response, lymphocyte-induced angiogenesis (LIA) elicited via the release of lymphokines from activated T cells. As angiogenic factors promote tumor development we investigated if SCS from tumor-bearing and tumor-resected mice contain factors capable of evoking a LIA response. Angiogenic activity was detected by SCS of small and large tumor-bearing mice as well as in large tumor-resected mice. On the other hand, SCS from small tumor-resected mice are not able to evoke an angiogenic response. Possibly, the large antigenic burden in LTRM stimulate the immune system in a different way than in STRM. We can suggest that the different behavior of spleen cells after small and large tumor removal can be explained by quantitative or qualitative changes in the spleen subpopulations.

Nature of the spleen cell populations capable of releasing tumor enhancing factor.

Supernatants obtained from short-cultured spleen cells (SCS) from BALB/c mice bearing a syngeneic mammary transplanted tumor--S13--showed enhancing activity on tumor growth when inoculated into the foot pad of normal syngeneic mice 24 hr before injection of S13 tumor cells. The present work was designed to characterize the spleen cell population responsible for the releasing of the enhancing factor (EF) as long as the tumor grows (small tumor bearing mice--STBM--and large tumor bearing mice--LTBM). Pretreatment of spleen cells with anti-Thy 1.2 serum + C' and nylon-wool columns were utilized to separate cell populations and to characterize the cellular source of the enhancing activity in the spleens of STBM and LTBM. In this tumor system, evidence is presented for distinct enhancing cell population operating in the spleens of STBM and LTBM. In early stages of tumor development, the EF was found to be associated with T and non-T cells, whereas in advanced stages of tumor growth, this activity was found to be associated with only T cells.

Regulation of tumor growth by soluble spleen factors: effect of tumor resection.

Supernatants of cultured spleen cells (SCS) from small-tumor-bearing mice (STBM) and large-tumor-bearing mice (LTBM) were tested in vivo by their ability to modify tumor development. We found that when inoculated with S13 tumor cells into the foot-pad of syngeneic normal BALB/c mice, SCS enhanced tumor growth as well as accelerated tumor takes. When STBM and LTBM were operated on, the persistence of the enhancing activity by SCS was found to be associated with tumor size at the time of tumor removal. As early as 24 hours after small-tumor resection, enhancing activity disappeared or diminished to undetectable levels. On the other hand, enhancing activity by SCS from large-tumor-resected mice (LTRM) persisted until 15 days after surgery, and then faded. Enhancing activity associated with SCS from S13-tumor-bearing mice (TBM) was also seen when injected with M3 cells, a syngeneic unrelated tumor. Normal SCS did not affect tumor development. It seems that the rate of disappearance of enhancing activity observed in SCS from S13-tumor-resected mice (TRM) is mainly dependent on the size of tumor burden at the time of surgery.

Immune reactivity of spleen cells in advanced stages of tumor growth.

In a syngeneic tumor model by an indirect migration inhibition technique, using spleen cells from mice bearing large tumors (ATB-SC) no detectable amounts of migration inhibition factor (MIF) was found in response to either tumor cells or tumor extract. Although lymphokine production was suppressed, ATB-SC were able to respond to exogenous MIF and also to transfer a positive delayed foot-pad reaction (DFR) when co-inoculated with both forms of antigen into normal mice. These observations showed that in advanced stages of tumor development, when the ability of spleen cells to produce MIF toward tumor cells or tumor extract is lost, neither their capability to transfer a positive DFR not their responsiveness to MIF-rich culture supernatants were impaired.

Idiotypic determinants in alloantigen receptors. I. Role in a leucocyte migration inhibition reaction.

An anti-idiotypic serum was prepared by injecting BALB anti-AKR serum into (BALB X AKR)F1 mice. Pretreatment of BALB anti-AKR immune cells with this anti-idiotypic serum plus complement abrogated a leucocyte migration inhibition reaction (LMIR) against AKR extracts but not against purified protein derivative By itself, the serum induced LMIR in immune cells but not in normal cells. The reaction was strain-specific and anti-Thy 1,2 plus complement sensitive. These results indicate that alloantigen receptors, able to trigger in LMIR in immune cells, have idiotypic determinants similar to those of circulating antibodies of the same specificity. Thus the LMIR is a good model for the study of receptors cell-mediated immune reactions of primed cells.